Bottom Line:
DRMs-associated IDE co-localized with Abeta and its distribution (DRMs vs. non-DRMs) and activity was sensitive to manipulation of lipid composition in vitro and in vivo.We detected a reduced amount of IDE in DRMs of membranes isolated from mice brain with endogenous reduced levels of cholesterol (Chol) due to targeted deletion of one seladin-1 allele.Our results support the notion that optimal substrate degradation by IDE may require its association with organized-DRMs. Alternatively, DRMs but not other plasma membrane regions, may act as platforms where Abeta accumulates, due to its hydrophobic properties, reaching local concentration close to its Km for IDE facilitating its clearance.

Background: Insulin degrading enzyme (IDE) is implicated in the regulation of amyloid beta (Abeta) steady-state levels in the brain, and its deficient expression and/or activity may be a risk factor in sporadic Alzheimer's disease (AD). Although IDE sub-cellular localization has been well studied, the compartments relevant to Abeta degradation remain to be determined.

Results: Our results of live immunofluorescence, immuno gold electron-microscopy and gradient fractionation concurred to the demonstration that endogenous IDE from brain tissues and cell cultures is, in addition to its other localizations, a detergent-resistant membrane (DRM)-associated metallopeptidase. Our pulse chase experiments were in accordance with the existence of two pools of IDE: the cytosolic one with a longer half-life and the membrane-IDE with a faster turn-over. DRMs-associated IDE co-localized with Abeta and its distribution (DRMs vs. non-DRMs) and activity was sensitive to manipulation of lipid composition in vitro and in vivo. When IDE was mis-located from DRMs by treating cells with methyl-beta-cyclodextrin (MbetaCD), endogenous Abeta accumulated in the extracellular space and exogenous Abeta proteolysis was impaired. We detected a reduced amount of IDE in DRMs of membranes isolated from mice brain with endogenous reduced levels of cholesterol (Chol) due to targeted deletion of one seladin-1 allele. We confirmed that a moderate shift of IDE from DRMs induced a substantial decrement on IDE-mediated insulin and Abeta degradation in vitro.

Conclusion: Our results support the notion that optimal substrate degradation by IDE may require its association with organized-DRMs. Alternatively, DRMs but not other plasma membrane regions, may act as platforms where Abeta accumulates, due to its hydrophobic properties, reaching local concentration close to its Km for IDE facilitating its clearance. Structural integrity of DRMs may also be required to tightly retain insulin receptor and IDE for insulin proteolysis. The concept that mis-location of Abeta degrading proteases away from DRMs may impair the physiological turn-over of Abeta in vivo deserves further investigation in light of therapeutic strategies based on enhancing Abeta proteolysis in which DRM protease-targeting may need to be taken into account.

Mentions:
Taking into account that des-localization of a given protein within a DRM as a consequence of lipid manipulations strongly suggests lipid raft association in vivo, we determined the effect of Chol, sphingolipids (SL) and phosphatitylcholine (PC) removalupon the association of IDE with DRMs. N2aWT cells were treated with MÎ²CD under experimental conditions reported to be "mild", without associated cytotoxicity [30]. Membrane fractions from control and MÎ²CD-treated cells showed similar amounts of Chol determined by an analytical procedure (1.26 Î¼g/mg vs. 1.46 Î¼g/mg) and similar amounts of IDE and flotillin (Fig. 5A) with a sharp reduction in insulin degradation from MÎ²CD-treated cells which was statistically significant as compared to control cells (28.6 Â± 8.5% vs. 66.9 Â± 1.5%; n = 3; p < 0.05) (Fig. 5B). To find a biochemical explanation for these results we determined the membrane lipid composition from control and MÎ²CD-treated cells by thin-liquid chromatography (TLC). Our data showed a decrement in the percentage of PC and SL in membranes from MÎ²CD-treated as compared to control cells (30% and 2.39% vs. 44% and 4.25%). In addition, we showed lipid-dependent changes in IDE- and flotillin-DRM association (Fig. 6A, framed region). In this regard, DRMs (fraction 3 and 4) from untreated cells (Fig. 6B upper panel, black bar) retain 12.6 Â± 0.6% of total membrane-associated IDE and 45.2 Â± 1.1% of total membrane-associated flotillin (Fig. 6B lower panel, black bar) while no reactivity for IDE was detected in DRMs of MÎ²CD-treated cells (Fig. 6B upper panel, white bar) and only a 32.5 Â± 0.6% (n = 3; p < 0.05) of flotillin reactivity was observed (Fig. 6B, lower panel, white bar). Lipid manipulations induced a rise of IDE immunoreactivity in "intermediate fractions" (fractions 6â€“8) from 47.4 Â± 2.3% to 66.2 Â± 1.7%.

Mentions:
Taking into account that des-localization of a given protein within a DRM as a consequence of lipid manipulations strongly suggests lipid raft association in vivo, we determined the effect of Chol, sphingolipids (SL) and phosphatitylcholine (PC) removalupon the association of IDE with DRMs. N2aWT cells were treated with MÎ²CD under experimental conditions reported to be "mild", without associated cytotoxicity [30]. Membrane fractions from control and MÎ²CD-treated cells showed similar amounts of Chol determined by an analytical procedure (1.26 Î¼g/mg vs. 1.46 Î¼g/mg) and similar amounts of IDE and flotillin (Fig. 5A) with a sharp reduction in insulin degradation from MÎ²CD-treated cells which was statistically significant as compared to control cells (28.6 Â± 8.5% vs. 66.9 Â± 1.5%; n = 3; p < 0.05) (Fig. 5B). To find a biochemical explanation for these results we determined the membrane lipid composition from control and MÎ²CD-treated cells by thin-liquid chromatography (TLC). Our data showed a decrement in the percentage of PC and SL in membranes from MÎ²CD-treated as compared to control cells (30% and 2.39% vs. 44% and 4.25%). In addition, we showed lipid-dependent changes in IDE- and flotillin-DRM association (Fig. 6A, framed region). In this regard, DRMs (fraction 3 and 4) from untreated cells (Fig. 6B upper panel, black bar) retain 12.6 Â± 0.6% of total membrane-associated IDE and 45.2 Â± 1.1% of total membrane-associated flotillin (Fig. 6B lower panel, black bar) while no reactivity for IDE was detected in DRMs of MÎ²CD-treated cells (Fig. 6B upper panel, white bar) and only a 32.5 Â± 0.6% (n = 3; p < 0.05) of flotillin reactivity was observed (Fig. 6B, lower panel, white bar). Lipid manipulations induced a rise of IDE immunoreactivity in "intermediate fractions" (fractions 6â€“8) from 47.4 Â± 2.3% to 66.2 Â± 1.7%.

Bottom Line:
DRMs-associated IDE co-localized with Abeta and its distribution (DRMs vs. non-DRMs) and activity was sensitive to manipulation of lipid composition in vitro and in vivo.We detected a reduced amount of IDE in DRMs of membranes isolated from mice brain with endogenous reduced levels of cholesterol (Chol) due to targeted deletion of one seladin-1 allele.Our results support the notion that optimal substrate degradation by IDE may require its association with organized-DRMs. Alternatively, DRMs but not other plasma membrane regions, may act as platforms where Abeta accumulates, due to its hydrophobic properties, reaching local concentration close to its Km for IDE facilitating its clearance.

Background: Insulin degrading enzyme (IDE) is implicated in the regulation of amyloid beta (Abeta) steady-state levels in the brain, and its deficient expression and/or activity may be a risk factor in sporadic Alzheimer's disease (AD). Although IDE sub-cellular localization has been well studied, the compartments relevant to Abeta degradation remain to be determined.

Results: Our results of live immunofluorescence, immuno gold electron-microscopy and gradient fractionation concurred to the demonstration that endogenous IDE from brain tissues and cell cultures is, in addition to its other localizations, a detergent-resistant membrane (DRM)-associated metallopeptidase. Our pulse chase experiments were in accordance with the existence of two pools of IDE: the cytosolic one with a longer half-life and the membrane-IDE with a faster turn-over. DRMs-associated IDE co-localized with Abeta and its distribution (DRMs vs. non-DRMs) and activity was sensitive to manipulation of lipid composition in vitro and in vivo. When IDE was mis-located from DRMs by treating cells with methyl-beta-cyclodextrin (MbetaCD), endogenous Abeta accumulated in the extracellular space and exogenous Abeta proteolysis was impaired. We detected a reduced amount of IDE in DRMs of membranes isolated from mice brain with endogenous reduced levels of cholesterol (Chol) due to targeted deletion of one seladin-1 allele. We confirmed that a moderate shift of IDE from DRMs induced a substantial decrement on IDE-mediated insulin and Abeta degradation in vitro.

Conclusion: Our results support the notion that optimal substrate degradation by IDE may require its association with organized-DRMs. Alternatively, DRMs but not other plasma membrane regions, may act as platforms where Abeta accumulates, due to its hydrophobic properties, reaching local concentration close to its Km for IDE facilitating its clearance. Structural integrity of DRMs may also be required to tightly retain insulin receptor and IDE for insulin proteolysis. The concept that mis-location of Abeta degrading proteases away from DRMs may impair the physiological turn-over of Abeta in vivo deserves further investigation in light of therapeutic strategies based on enhancing Abeta proteolysis in which DRM protease-targeting may need to be taken into account.